Identification of a SmD3 epitope with a single symmetrical dimethylation of an arginine residue as a specific target of a subpopulation of anti-Sm antibodies.

Mahler M, Fritzler MJ, Blüthner M - Arthritis Res. Ther. (2004)

Bottom Line:
Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population.In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide.These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients.

ABSTRACTAnti-Sm antibodies, identified in 1966 by Tan and Kunkel, are highly specific serological markers for systemic lupus erythrematosus (SLE). Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population. The Sm autoantigen complex comprises at least nine different polypeptides. All of these core proteins can serve as targets of the anti-Sm B-cell response, but most frequently the B and D polypeptides are involved. Because the BB'Sm proteins share cross-reactive epitopes (PPPGMRPP) with U1 specific ribonucleoproteins, which are more frequently targeted by antibodies that are present in patients with mixed connective tissue disease, the SmD polypeptides are regarded as the Sm autoantigens that are most specific to SLE. It was recently shown that the polypeptides D1, D3 and BB' contain symmetrical dimethylarginine, which is a component of a major autoepitope within the carboxyl-terminus of SmD1. In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide. Using immobilized peptides, we confirmed that the dimethylated arginine residues play an essential role in the formation of major SmD1 and SmD3 autoepitopes. Moreover, we demonstrated that one particular peptide of SmD3 represents a more sensitive and more reliable substrate for the detection of a subclass of anti-Sm antibodies. Twenty-eight out of 176 (15.9%) SLE patients but only one out of 449 (0.2%) control individuals tested positive for the anti-SmD3 peptide (SMP) antibodies in a new ELISA system. These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients. Thus, anti-SMP detection using ELISA represents a new serological marker with which to diagnose and discriminate between systemic autoimmune disorders.

Figure 1: Epitope analysis of SmD1 and SmD3. Carboxyl-terminal regions of (a) SmD1 and (b) SmD3 were synthesized as peptide arrays (15 mers; two amino acids offset) and probed with patient sera. Each arginine containing peptide was synthesized as three variants, one with natural arginine (R), one with symmetrical dimethylarginine (sDMA) and one with asymmetrical dimethylarginine (asDMA) at the respective positions. In addition, a highly reactive SmD3 peptide was synthesized with certain combinations of natural arginine and sDMA. A significant effect of dimethylation of arginine residues on the antigenicity of SmD derived peptides was observed (black squares indicate strong reactivity; white indicate no reactivity). Binding of an anti-Sm negative serum sample (Varelisa® Sm) that contained anti-centromere antibodies (ACA) could be observed with SmD1 but not with SmD3 peptides. Thus, the immunoreactive peptide no. 77 was further tested in (c) a replacement experiment. The SmD3 peptide exhibited exclusive reactivity with the Sm-positive sera.

Mentions:
To evaluate the effect of arginine dimethylation on the antigenicity of SmD1 and SmD3 and to localize relevant epitopes on both polypeptides, a panel of anti-Sm sera was tested for reactivity with peptide arrays (15 mer, two offset) covering the carboxyl-terminal region of SmD1 (P13641) and SmD3 (P43331). The results show that dimethylation of arginine residues significantly affects the binding of anti-Sm antibodies to carboxyl-terminal SmD1 and SmD3 peptides (Fig. 1). All anti-Sm sera exhibited increased binding to SmD1 peptides containing sDMA as compared with those containing unmethylated arginine (Fig. 1a). In particular, peptides that exclusively consist of glycine and sDMA repeats exhibited strong reactivity with the antibodies (peptide nos 9, 10 and 11). Nevertheless, SmD1 peptides containing sDMA represent a rather unspecific substrate for anti-Sm antibodies because they were also bound by sera that contained anti-centromere antibodies. Interestingly, those anti-centromere antibodies also bound to peptides containing the asymmetrical form of DMA but to a lesser extent.

Figure 1: Epitope analysis of SmD1 and SmD3. Carboxyl-terminal regions of (a) SmD1 and (b) SmD3 were synthesized as peptide arrays (15 mers; two amino acids offset) and probed with patient sera. Each arginine containing peptide was synthesized as three variants, one with natural arginine (R), one with symmetrical dimethylarginine (sDMA) and one with asymmetrical dimethylarginine (asDMA) at the respective positions. In addition, a highly reactive SmD3 peptide was synthesized with certain combinations of natural arginine and sDMA. A significant effect of dimethylation of arginine residues on the antigenicity of SmD derived peptides was observed (black squares indicate strong reactivity; white indicate no reactivity). Binding of an anti-Sm negative serum sample (Varelisa® Sm) that contained anti-centromere antibodies (ACA) could be observed with SmD1 but not with SmD3 peptides. Thus, the immunoreactive peptide no. 77 was further tested in (c) a replacement experiment. The SmD3 peptide exhibited exclusive reactivity with the Sm-positive sera.

Mentions:
To evaluate the effect of arginine dimethylation on the antigenicity of SmD1 and SmD3 and to localize relevant epitopes on both polypeptides, a panel of anti-Sm sera was tested for reactivity with peptide arrays (15 mer, two offset) covering the carboxyl-terminal region of SmD1 (P13641) and SmD3 (P43331). The results show that dimethylation of arginine residues significantly affects the binding of anti-Sm antibodies to carboxyl-terminal SmD1 and SmD3 peptides (Fig. 1). All anti-Sm sera exhibited increased binding to SmD1 peptides containing sDMA as compared with those containing unmethylated arginine (Fig. 1a). In particular, peptides that exclusively consist of glycine and sDMA repeats exhibited strong reactivity with the antibodies (peptide nos 9, 10 and 11). Nevertheless, SmD1 peptides containing sDMA represent a rather unspecific substrate for anti-Sm antibodies because they were also bound by sera that contained anti-centromere antibodies. Interestingly, those anti-centromere antibodies also bound to peptides containing the asymmetrical form of DMA but to a lesser extent.

Bottom Line:
Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population.In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide.These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients.

ABSTRACTAnti-Sm antibodies, identified in 1966 by Tan and Kunkel, are highly specific serological markers for systemic lupus erythrematosus (SLE). Anti-Sm reactivity is found in 5-30% of SLE patients, depending on the autoantibody detection system and the racial background of the SLE population. The Sm autoantigen complex comprises at least nine different polypeptides. All of these core proteins can serve as targets of the anti-Sm B-cell response, but most frequently the B and D polypeptides are involved. Because the BB'Sm proteins share cross-reactive epitopes (PPPGMRPP) with U1 specific ribonucleoproteins, which are more frequently targeted by antibodies that are present in patients with mixed connective tissue disease, the SmD polypeptides are regarded as the Sm autoantigens that are most specific to SLE. It was recently shown that the polypeptides D1, D3 and BB' contain symmetrical dimethylarginine, which is a component of a major autoepitope within the carboxyl-terminus of SmD1. In one of those studies, a synthetic dimethylated peptide of SmD1 (amino acids 95-119) exhibited significantly increased immunoreactivity as compared with unmodified SmD1 peptide. Using immobilized peptides, we confirmed that the dimethylated arginine residues play an essential role in the formation of major SmD1 and SmD3 autoepitopes. Moreover, we demonstrated that one particular peptide of SmD3 represents a more sensitive and more reliable substrate for the detection of a subclass of anti-Sm antibodies. Twenty-eight out of 176 (15.9%) SLE patients but only one out of 449 (0.2%) control individuals tested positive for the anti-SmD3 peptide (SMP) antibodies in a new ELISA system. These data indicate that anti-SMP antibodies are exclusively present in sera from SLE patients. Thus, anti-SMP detection using ELISA represents a new serological marker with which to diagnose and discriminate between systemic autoimmune disorders.